Antibiotics

ABSTRACT

The invention provides compounds of the general formula: 
                         
wherein
         X is selected from the group consisting of oxygen, sulfur and nitrogen-containing groups selected from azine, oxime, hydrazone, aromatic hydrazone, aliphatic hydrazone, semicarbazone, guanidinyl group, and aliphatic or aromatic imines;   Y is selected from the group of halogens;   R 1  and R 2  are independently selected from the group consisting of H, lower alkyl group (of C 1  up to C 6 ), lower hydroxyalkyl group (of C 1  up to C 6 ), lower O-alkyl group (of C 1  up to C 6 ), lower alkylcarbonyl group (of C 2  up to C 6 ), lower alkyloxycarbonyl group (of C 1  up to C 6 ), haloalkylcarbonyl group (of C 1  up to C 6 ), or arylsulfonyl group, and their pharmaceutically acceptable salts.       
     The compounds are useful as active ingredients in pharmaceutical compositions. The invention relates to the use of the said compounds as a medicament, in particular as an antibiotic.

The present application is directed to a new class of compounds whichare useful active ingredients in pharmaceutical compositions, inparticular as antibiotics.

Although a large variety of antibiotics, i.e. antibacterial agents, areavailable in the prior art, there is a continuous need for thedevelopment of new substances with this activity, in particular as manybacteria have developed resistances against known antibiotics.

From the prior art, ciprofloxacin(1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinolinecarboxylicacid) is known to be an effective antibacterial agent acting as a gyraseinhibitor. Other derivatives of 4-quinolinone-3-carboxylic acid, such asnorfloxacin and of loxacin, are also known to be active as antibiotics.For example, DE 35 25 108 A1 discloses a number of antibacteriallyactive quinolone carboxylic esters.

M. Reuman, M. A. Eissenstat and J. D. Weaver III “Cyanide MediatedDecarboxylation of 1-Substituted-4-oxoquinoline and4-oxo-1,8-naphthyridine-3-carboxylic Acids”, Tetrahedron Letters, Vol.35, No. 45, pp. 8303–8306 (1994) discloses the decarboxylation of4-oxo-3-quinolinecarboxylic acids. However, no specific pharmaceuticalactivity is disclosed for such decarboxylated compounds.

H. Kondo, F. Sakamoto, K. Kawakami and G. Tsukamoto “Studies onProdrugs. 7. Synthesis and Antimicrobial Activity of 3-FormylquinoloneDerivatives”, J. Med. Chem. 1988, 31, pp. 221–225 discloses 3-formylquinolone derivatives.

WO 93/15084 is directed to novel cephalosporin compounds, disclosing, asintermediates of the synthesis thereof, specific quinolone derivatives.

Surprisingly, it has now been found that a group of compounds beingstructurally related to this known group of compounds such asciprofloxacin and the like, but without the carboxylic acid or esterfunctionality, common to all such known active substances, are not onlyalso highly effective as antibiotics, but even show a superiorantibacterial effect.

Thus, this invention is related to a new class of compounds having thegeneral formula (I)

wherein

-   X is selected from the group consisting of oxygen, sulfur and    nitrogen-containing groups selected from azine, oxime, hydrazone,    aromatic hydrazone, aliphatic hydrazone, semicarbazone, guanidinyl    group, or aliphatic or aromatic imines;-   Y is selected from the group of halogens;-   R₁ and R₂ are independently selected from the group consisting of H,    lower alkyl group (of C₁ up to C₆), lower hydroxyalkyl group (of C₁    up to C₆), lower O-alkyl group (of C₁ up to C₆), lower alkylcarbonyl    group (of C₂ up to C₆), lower alkyloxycarbonyl group (of C₁ up to    C₆), haloalkylcarbonyl group (of C₁ up to C₆), or arylsulfonyl    group,-   and their pharmaceutically acceptable salts,    with the exception of a compound according to formula (I) with X    being sulfur, R₁ being hydrogen and R₂ being either hydrogen or    methyl, and a compound according to formula (I) with X being O, R₁    being hydrogen, and R₂ being hydrogen or ethyl.

A preferred group are the fluoroquinolinones, i.e. with X being oxygenand Y being fluorine. The presently most preferred compound is1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7(1-piperazinyl)-quinoline,referred to as compound (A).

Suitable salts of the compounds according to formula (I) are those ofinorganic and organic acids, e.g. hydrochloric acid, sulphuric acid,phosphoric acid, citric acid, malic acid, stearic acid, succinic acid,lactic acid, aspartic acid, glutamic acid, gluconic acid, acetic acid,and formic acid as examples; other acids are understood to be suitableas well.

The present invention is also related to a pharmaceutical compositioncomprising a compound having the general formula (I)

-   -   wherein    -   X is selected from the group consisting of oxygen, sulfur and        nitrogen-containing groups selected from azine, oxime,        hydrazone, aromatic hydrazone, aliphatic hydrazone,        semicarbazone, guanidinyl group, or aliphatic or aromatic        imines;    -   Y is selected from the group of halogens;    -   R₁ and R₂ are independently selected from the group consisting        of H, lower alkyl group (of C₁ up to C₆), lower hydroxyalkyl        group (of C₁ up to C₆), lower O-alkyl group (of C₁ up to C₆),        lower alkylcarbonyl group (of C₂ up to C₆), lower        alkyloxycarbonyl group (of C₁ up to C₆), haloalkylcarbonyl group        (of C₁ up to C₆), or arylsulfonyl group,        or a pharmaceutically acceptable salt thereof, as active        ingredient together with pharmaceutically acceptable adjuvants,        diluents, excepients or carriers, in all known pharmaceutical        dosage forms.

Furthermore, the present invention is related to a compound having thegeneral formula (I)

-   -   wherein    -   X is selected from the group consisting of oxygen, sulfur and        nitrogen-containing groups selected from azine, oxime,        hydrazone, aromatic hydrazone, aliphatic hydrazone,        semicarbazone, guanidinyl group, or aliphatic or aromatic        imines;    -   Y is selected from the group of halogens;    -   R₁ and R₂ are independently selected from the group consisting        of H, lower alkyl group (of C₁ up to C₆), lower hydroxyalkyl        group (of C₁ up to C₆), lower O-alkyl group (of C₁ up to C₆),        lower alkylcarbonyl group (of C₂ up to C₆), lower        alkyloxycarbonyl group (of C₁ up to C₆), haloalkylcarbonyl group        (of C₁ up to C₆), or arylsulfonyl group,        or a pharmaceutically acceptable salt thereof or the composition        according to claim 6 for use as a medicament for the treatment        of humans and animals.

Finally, the invention is directed to the use of a compound having thegeneral formula (I)

-   -   wherein    -   X is selected from the group consisting of oxygen, sulfur and        nitrogen-containing groups selected from azine, oxime,        hydrazone, aromatic hydrazone, aliphatic hydrazone,        semicarbazone, guanidinyl group, or aliphatic or aromatic        imines;    -   Y is selected from the group of halogens;    -   R₁ and R₂ are independently selected from the group consisting        of H, lower alkyl group (of C₁ up to C₆), lower hydroxyalkyl        group (of C₁ up to C₆), lower O-alkyl group (of C₁ up to C₆),        lower alkylcarbonyl group (of C₂ up to C₆), lower        alkyloxycarbonyl group (of C₁ up to C₆), haloalkylcarbonyl group        (of C₁ up to C₆), or arylsulfonyl group,        or a pharmaceutically acceptable salt thereof or of a        composition according to claim 6 as an antibiotic.

Experimental Procedure:

The initial starting material used in this invention for the preparationof all the example compounds according to formula (I) specified in theExamples is ciprofloxacin (where R₁═COOH; R₂═H; Y═F; X═O). Compound (A)is prepared from ciprofloxacin as follows:

A mixture of ciprofloxacin (5.0 g) and formaldehyde solution (25 ml) inn-butanol (50 ml) was refluxed for 3 hours. The solvent was evaporated.The precipitated solid was crystallized from methanol or ethanol ascolourless leaflet crystals to give1-cyclopropyl-6-fluoro-1,4-dihydro-4-oyo-7(1-piperazinyl)-quinoline,referred to as compound (A) in this invention. It has a m.p. of188–190°, with a yield of 4.1 g, representing 94.5% percent yield fromthe starting materials. Compound (A) could be prepared under the sameexperimental conditions using other alcohols with a lower alkyl groupfrom C₁ to C₅.

The hydrochloride salt of this compound was prepared by stirring (5.0 g)of compound (A) in ethanol (15 ml) and the pH of the solution wasadjusted to pH 2.7–3.2 using hydrochloric acid solution. Theprecipitated white product was separated by filtration, dried at 65° C.It has a m.p. with decomposition at 321–323°. Compound (A) was confirmedusing several spectroscopic techniques as follows:

IR (KBr, cm⁻¹): 1257, 1471, 1500, 1629, 1757; MS: M⁺(287), (M⁺-C₂H₄N),(M⁺-C₃H₇N), (M⁺-C₄H₉N); NMR:

The salts of the preferred embodiment of the invention compound (A) wereprepared by digesting compound (A) with alcohol of lower alkyl groupfrom C₁ to C₃. The pH of the solution is adjusted to pH between 2.0–5.0with maximum yield at pH of 2.8–3.0 with the relevantly used acid toadjust the pH. The resulting salt may contain varying amount of water,which can be adjusted to be one mole of water in the resulting salt bydrying the salt at a temperature ranging from 60–70° C.

The hydrochloride salt of the said compound (A) is stable and moresoluble in water than ciprofloxacin hydrochloride. It gave a betterantibacterial effect towards gram-positive and gram-negative organismsthan ciprofloxacin hydrochloride when both compounds are tested underthe same experimental conditions.

Another preferred embodiment of the invention is1-cyclopropyl-6-fluoro-7-methylpiperazino-4-oxo-1,4-dihydroquinolinewill be referred to as Compound (B) of this invention, which is formedin good yield by reacting compound (A) with aliphatic aldehydes of loweralkyl group of C₁ to C₆. The hydrochloride salt of Compound (B) wasobtained in then same method used for compound (A) salt preparation.Compound (B) hydrochloride salt gave better antibacterial effect towardsgram-positive and gram-negative organisms than ciprofloxacinhydrochloride. Other derivatives from compound (A) were also prepared asshown in the examples of this invention.

The following examples are cited to explain the invention but not tolimit the scope of the invention in any way.

EXAMPLE 11-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-(4-methyl)piperazinyl)-quinoline

5.0 g of compound (A) was refluxed with 20 ml formaldehyde solution for3 hours. The separated solid was crystallized from ethanol to give thecorresponding1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-(4-methyl)piperazinyl)-quinoline,referred to as compound (B), in yield of 4.5 g as colourless crystals.It has m.p. 238–240°. The hydrochloride salt of compound (B) wasprepared by the same method used for the preparation of thehydrochloride salt of compound (A).

IR spectrum of salt B (KBr, cm⁻¹): 1271, 1471, 1643, 1743. MS: M⁺ 301.

EXAMPLE 21-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-(4-chloroacetyl)-piperazinyl)-quinoline

A solution of chloroacetyl chloride (2.5 ml) in acetone (10 ml) wasadded with stirring to a solution of1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-quinoline (A)(5.0 g) in acetone (20 ml) containing pyridine (5 ml) during 15 min. Theprecipitated product was separated by filtration to give thecorresponding1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-(4-chloroacetyl)-piperazinyl)-quinolineas colourless crystals, m.p. 262–64°. Its hydrochloride salt has amelting point with decomposition at 309–310° C.

IR spectrum of salt (KBr, cm⁻¹): 1273, 1350, 1480, 1500, 1630, 1730.

EXAMPLE 31-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-4-methoxycarbonyl)-piperazinyl)-quinoline

The title compound was synthesized from compound (A) by a procedureanalogous to that method mentioned under example 2 using methylchloroformate instead of chloroacetyl chloride. The product wasseparated as colourless crystals with m.p. 206–207° C.

IR spectrum (KBr, cm⁻¹): 1275, 1300, 1380, 1480, 1500, 1635, 1730, 1750.

EXAMPLE 41-cyclopropyl-6-fluoro-1,4-dihydro-4-hydrazono-7-(1-piperazinyl)-quinoline

A mixture of 2.0 g of compound (A) and 4 ml hydrazine hydrate in 15 mlethanol was refluxed on water-bath for 2 hours. The precipitated solidwas separated by filtration. The product was re-crystallized fromethanol to give colourless crystals with m.p. 270–272° C.

IR spectrum (KBr, cm⁻¹): 1270, 1320, 1380, 1480, 1630, 3400 (broad); MS:301(M⁺)

EXAMPLE 51-cyclopropyl-6-fluoro-1,4-dihydro-4-guanidino-7-(1-piperazinyl)-quinoline

A solution of guanidine hydrochloride (1.5 g) in 10 ml water was addedto a solution of compound (A) in ethanol (20 ml) containing (1.0 g)sodium carbonate. The mixture was refluxed for 3 hours. The precipitatedsolid was separated by filtration and had a m.p. 248–250° C.

IR spectrum (KBr, cm⁻¹): 1270, 1320, 1390, 1480, 1629, 3400 (broad)

EXAMPLE 61-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-(4-p-tosyl)-piperazinyl)-quinoline

The title compound was prepared from compound (A) by a procedure similarto that method mentioned under example 2 using p-tolylsulfonyl chloride(p-tosyl chloride). The product was separated as pale yellow crystalswith m.p. 264–266° C.

IR spectrum (KBr, cm⁻¹): 1270, 1350, 1475, 1495, 1510, 1630, 1730

Antimicrobial Activity of the Compounds of the Present Invention:

An aqueous solution of the salts of the compounds reported in thisinvention namely compound (A) and compound (B) were tested in vitroagainst E. coli and St. aureus according to an established clinicalprocedure (Mackie and McCartney: Practical Medical Microbiology,Churchill livingstone, 1996, 14^(th) edition, p 159–163). The resultsshow that these compounds gave better antibacterial effect againstgram-positive and gram-negative organisms than cipro-floxacin-HCl as areference compound. Table (1) below shows the minimum inhibitoryconcentrations (MIC) of compound (A) compared to ciprofloxacin in theirhydrochloride salt.

TABLE 1 Minimum Inhibitory Concentration (MIC) Tested compound E. coliSt. aureus Ciprofloxacin HCl salt 0.02 μg/ml 0.15 μg/ml Compound (A) HClsalt 0.01 μg/ml 0.08 μg/ml

The minimum inhibitory concentration (MIC) of compound (A) andciprofloxacin were also studied using clinically isolated microorganismstaphylococcus aureus and pseudomotias aeruginose.

Table (2) reports the effective maintenance dose of the compounds of theinvention necessary to control bacterial re-growth. The experimentalwork was carried out according to an established clinical procedure onmice (Mackie and McCartney, ibid). The results obtained show thatcompound (A) is more sensitive and effective as antibacterial agentsthan ciprofloxacin hydrochloride. This fact was elucidated after furtherincubation of the positive samples for up to 16 hours for both compoundsin which, the results show that, growth rate of bacteria in the samplerelated to ciprofloxacin hydrochloride increased with time, whilecompound (A) showed very slow bacterial growth at a dose of 0.0 μg/ml.This reflects the ability of compound (A) to virtually inhibiting thegrowth of bacteria compared to ciprofloxacin under the same experimentalconditions as stated in this text.

TABLE 2 Micro organism Staphylococcus Pseudomones Material aureusaeruginose Ciprofloxacin HCl salt 0.08 μg/ml 0.06 μg/ml Compound (A) HCl0.06 μg/ml 0.05 μg/ml

LD₅₀ for both compounds were also studied on mice according to anestablished clinical procedure (Mackie and McCartney, ibid) and theresults are reported in table (3).

TABLE 3 LD₅₀ for the compounds of the invention Compound LD₅₀Ciprofloxacin HCl salt  8 mg/kg mice Compound (A), HCl salt 28 mg/kgmice

The features disclosed in the foregoing description and in the claimsmay both separately and in any combination thereof, be material forrealizing the invention in diverse forms thereof.

1. A compound having the formula (I)

wherein X is oxygen, a hydrazone group, or a guanidinyl group; Y is ahalogen; R₁ is selected from the group consisting of H, a C₁–C₆ alkylgroup, a C₁–C₆ hydroxyalkyl group, a C₁–C₆ O-alkyl group, a C₂–C₆alkylcarbonyl group, a C₁–C₆ haloalkylcarbonyl group, or an arylsulfonylgroup; and R₂ is selected from the group consisting of H, a C₁–C₆ alkylgroup, a C₁–C₆ hydroxyalkyl group, a C₁–C₆ O-alkyl group, a C₂–C₆alkylcarbonyl group, a C₁–C₆ alkyloxycarbonyl group, a C₁–C₆haloalkylcarbonyl group, or an arylsulfonyl group; or pharmaceuticallyacceptable salt thereof, with the proviso that the compound is otherthan a compound of formula (I) where X is O, R₁ is H, and R₂ is H orethyl.
 2. The compound according to of claim 1, wherein X is oxygen. 3.The compound according to of claim 1, wherein Y is fluorine.
 4. Thecompound according to claim 1 wherein R₁ and R₂ are independentlyselected from H or a C₁–C₆ alkyl group.
 5. The compound according toclaim 4, wherein both R₁ and R₂ are H.
 6. The compound1-cyclopropyl-6-fluoro-7-methypiperazino4-oxo-1,4- dihydroquinoline orpharmaceutically acceptable salts thereof.
 7. The compound1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-(4-chloroacetyl)-piperazinyl)-quinolineor pharmaceutically acceptable salts thereof.
 8. The compound1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-(4-methoxycarbonyl)-piperazinyl)-quinolineor pharmaceutically acceptable salts thereof.
 9. The compound1-cyclopropyl-6-fluoro-1,4-dihydro-4-hydrazine-7-(1-piperazinyl)-quinolineor pharmaceutically acceptable salts thereof.
 10. The compound1-cyclopropyl-6-fluoro-1,4-dihydro-4-guanidino-7-(1-piperazinyl)-quinolineor pharmaceutically acceptable salts thereof.
 11. A pharmaceuticalcomposition comprising a compound of claim 1, as active ingredient; anda pharmaceutically acceptable adjuvant, diluent, excipient or carrier.12. An antimicrobial composition which comprises an effective amount of:1-cyclopropyl-6-fluoro-7-methypiperazino-4-oxo-1,4-dihydroquinoline,1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-(4-chloroacetyl)-piperazinyl)-quinoline,1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-(4-methoxycarbonyl)-piperazinyl)-quinoline,1-cyclopropyl-6-fluoro-1,4-dihydro-4-hydrazine-7-(1-piperazinyl)-quinoline,1-cyclopropyl-6-fluoro-1,4-dihydro-4-guanidino-7-(1-piperazinyl)-quinoline,or combinations thereof, and a pharmaceutically acceptable adjuvant,diluent, excipient or carrier.
 13. A method for treating bacterialinfection in a subject comprising administering an antibacteriallyeffective amount of a compound of the formula presented in claim
 1. 14.A method for treating bacterial infection in a subject comprisingadministering an antibacterially effective amount of a pharmaceuticalcomposition of claim
 11. 15. A method for inhibiting bacterial growthcomprising administering to the bacteria an effective around of acompound of the formula presented in claim
 1. 16. The method of claim15, wherein the bacterial growth is in vitro.
 17. The method of claim15, wherein the bacterial growth is in vivo.